Gel microdrops in genetic analysis

ABSTRACT

The invention provides methods of nucleic acid analysis. Such methods entail forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity. The population of gel microdrops is then contacted with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop. At least one gel microdrop is then analyzed or detected. The biological entities can be cells, viruses, nuclei and chromosomes.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.10/428,912, filed May 2, 2003, which is a divisional of U.S. applicationSer. No. 09/369,640, filed Aug. 6, 1999, now U.S. Pat. No. 6,586,176,which claims priority from U.S. Application No. 60/095,721, filed Aug.7, 1998, the disclosures of which are incorporated by reference in theirentirety for all purposes.

TECHNICAL FIELD

The present invention resides in the field of genetic analysis.

BACKGROUND

Cytogenetic testing is still in its infancy. Current cytogenetic methodsare limited to analysis of gene aberrations easily detectable in cells,nuclei, or chromosomes, in part, because slide based methods are highlymanual. Analysis of aberrations present in low frequency is notroutinely performed in the clinical setting because many slides ofcells, nuclei or chromosomes would have to be evaluated to establishstatistical frequency.

Banding is the classical approach used for analyzing chromosomes inmetaphase spreads. This method is based on staining which results indark bands in the region of the chromosome where the chromatin occurs athigher density. The banding pattern is specific for each chromosome andallows identification for karyotyping, which is the determination ofeach chromosome's copy number. However, banding resolution is notsufficient to detect small deletions or additions of chromosomal mass,which occur in a variety of disease conditions, particularly in cancers.

Fluorescent in situ hybridization is another approach used to localizegenomic DNA fragments or to paint whole chromosomes and to detect andcharacterize genetic abnormalities including translocations (31, 40),aneusomy (41, 42), and gene amplification (43). These geneticabnormalities can be detected in individual cells, chromosomes, ornuclei to assess of tumor genotype, analyze genetic heterogeneity, anddetect malignant cells. To preserve integrity in FISH applications,chromosomes are typically adsorbed onto glass slides for analysis.Analysis therefore requires microscopic evaluation of individual slideslimiting automation and rapid sample processing. Fluorescent in situhybridizations prepared on glass slides rely not only on the assay andreagents but on the instrumentation and the expertise and ingenuity ofthe scientists using it resulting in poor reproducibility. An inherentlimitation to this technology is that at least 100 kb of DNA sequence ina single cell must be present for detection (68-70). In addition, harshconditions for fixing either tissue or intact cells to a glass slide areless than optimal: up to 90% of the assay sample can be lost from theglass support.

Some chromosomes can also be resolved by fluorescent staining followedby flow cytometry (14,15). Successful chromosome sorting is, however,dependent on the binding characteristics of fluorescent dyes and theextent to which the chromosome of interest can be distinguished fromchromosomes of similar size, clumps of chromosomes, and debriscontaining DNA (13). Although this approach has resulted in theconstruction of yeast artificial chromosome (YAC) libraries for mappingstudies (16) in species which have chromosomes of similar size, such asmouse, arabidopsis, and 20% of the human chromosomes, unambiguousresolution has not been possible. Flow sorting based on dye uptake ispossible for well resolved chromosomes, but this method works poorly forchromosomes which are similar in size and base composition, mainly humanchromosomes 9-12 and the majority of mouse chromosomes. Furthermore,flow cytometry cannot currently be used to analyze hybridizedchromosomes prepared by conventional methods because unfixed chromosomesfall apart using high temperatures and/or formamide.

SUMMARY OF THE CLAIMED INVENTION

The invention provides methods of nucleic acid analysis. Such methodsentail forming a population of gel microdrops encapsulating a populationof biological entities, each entity comprising a nucleic acid, wherebyat least some microdrops in the population each encapsulate a singleentity. Nucleic acids can be DNA or RNA. The population of gelmicrodrops is then contacted with a probe under conditions whereby theprobe specifically hybridizes to at least one complementary sequence inthe nucleic acid in at least one gel microdrop. At least one gelmicrodrop is then analyzed or detected. The biological entities can becells, viruses, nuclei and chromosomes.

In some methods, at least 10,000 biological entities are encapsulated.In some methods, the biological entities are not fixed chemically beforethe contacting step. In some methods, nucleic acids are amplified beforethe contacting step. Suitable materials for forming droplets includeagarose, alginate, carrageenan, or polyacrylamide.

In some methods, nucleic acids are recovered from microdrops bydigestion with agarase. Optionally, the recovered DNA can be digestedwith a restriction enzyme with or without prior digestion of agarase. Insome methods, the gel matrix is crosslinked with itself and/or nucleicacid being analyzed, typically, between the denaturation and contactingsteps. In some method, the hybridization is performed at a temperatureof over 68° C. or in the presence of a formamide concentration greaterthan 20%. In some methods, the microdrops further comprise a reagentthat amplifies a signal from the labeled probe. For example, the probecan be labeled with an enzyme, and the reagent can be a substrate forthe enzyme.

In some methods, microdrops are isolated by FACS™. In some methods, thebiological entities are a population of chromosomes obtained from apopulation of different cells in a patient. In some methods, the ratioof a subpopulation of microdrops containing a chromosome hybridized tothe probe to a subpopulation of microdrops containing a chromosome nothybridized to the probe is determined. In some methods, the probehybridizes to a nucleic acid segment bearing a mutation and the ratioindicates the proportion of cells in the population bearing themutation. Such methods are particularly useful for analyzing somaticmutations.

In some methods, an isolated microdrop containing a single chromosome isused to prepare a single chromosomal fragment library. Such a librarycan in turn be used for preparing probes for a single chromosome, suchas painting or reverse painting probes.

Gel microdrops encapsulated biological entities can be stored before orafter the hybridization step for a period of at least six months.

The invention further provides methods of diagnosing a disease due to agenetic mutation. Such methods entail obtaining a sample of cells from apatient. A population of chromosomes from the sample in thenencapsulated in a population of microdrops. One then contacts themicrodrops with a first probe that is complementary to a nucleic acidsegment containing the somatic mutation, and a second probecomplementary to the chromosome in which the somatic mutation occurs ata site distal to the somatic mutation. The first probe hybridizes tomicrodrops bearing the chromosome with a somatic mutation and the secondprobe hybridizes to microdrops bearing the chromosome irrespectivewhether the somatic mutation is present. One then determines the ratioof microdrops hybridizing to the first probe and hybridizing to thesecond probe. The ration can then be used to diagnose the existence orprognosis of the disease from the ratio. Such methods are particularuseful for diagnosing existence or prognosis of cancer.

The invention further provides methods of chromosome analysis. Suchmethods entail forming a population of gel microdrops encapsulating apopulation of nucleic, whereby at least some microdrops in thepopulation each encapsulate a single nucleus. One then contacts thepopulation of gel microdrops with a probe under conditions whereby theprobe specifically hybridizes to at least one complementary sequence inat least one chromosome in a nucleus of least one gel microdrop. Onethen isolates or detects the at least one gel microdrop.

The invention further provides methods of isolating chromosomes. Somesuch methods entail culturing a population of cells in genistein andcolcemid to synchronize chromosomes in metaphase, and isolatingchromosomes from the cells. Other methods, which can be used inconjunction or independently of the previously described methods, entaillysing a population of cells to form a lysate. The lysate is thentreated with an antibody linked to a magnetic particles, wherein theantibody specifically binds to one or more chromosomes in the cells.Magnetic particles are then isolated from the lysate.

The invention further provides methods of chromosome analysis. Suchmethods entail forming a population of gel microdrops encapsulating apopulation of cells or nuclei, whereby at least some microdrops in thepopulation each encapsulate a single nucleus. One then contacts thepopulation of gel microdrops with a probe under conditions whereby theprobe specifically hybridizes to at least one complementary sequence inat least one nucleus in at least one gel microdrop. One then isolates ordetects the at least one gel microdrop.

The invention further provides a kit comprising high melting temperatureagarose, emulsification equipment, and a label indicating how to use thekit for probe hybridization analysis. Optionally, the kit also includesat least one probe that hybridizes to a nucleic acid.

DEFINITIONS

An isolated species means an object species invention that is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). Preferably, anisolated species comprises at least about 50, 80 or 90 percent (on amolar basis) of all macromolecular species present. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detectionmethods).

Polymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Apolymorphic marker or site is the locus at which divergence occurs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows encapsulated and unencapsulated human, mouse and plantchromosomes.

FIG. 2 shows gel electrophoresis of chromosomal DNA.

-   Unencapsulated, freshly isolated chromosomes: b-mouse, e-human    K-562, g-human REH; Unencapsulated chromosomes one month after    isolation: c-mouse, f-human K-562, h-human REH; Encapsulated    chromosomes stored for 6 months: d-mouse, i-human K-562, j-human    REH; lambda DNA (0.1 μg) used as a control: a,k.

FIG. 3A (4) shows flow sorting of human chromosome 22 followingmicrodrops in situ hybridization using a bcr locus specific probe. FIG.3B shows sorted chromosomes visualized using fluorescent microscopy.

FIG. 4 (5) shows purity of chromosome 22 after sorting measure by thepolymerase chain reaction using primer sets specific from chromosomes10, 21 or 22.

FIG. 5 (8) Restriction digestion of encapsulated chromosomes.

FIG. 6 (9), detection of translocated chromosome 22 left chromosome 9middle, and translocation-free chromosome 22 right by MISH are shown.

FIG. 7 shows flow cytometric detection of gag HIV RNA in encapsulatedHIV infected cells after hybridization with two HRP-labeled oligoprobes.

FIG. 8 shows detection of telomerase mRNA in HL-60 (model cancer cellline) and human PBMCs using fluorescein-labeled oligonucleotide probes.

DETAILED DESCRIPTION I. General

The invention provides methods of analyzing populations of nucleic-acidcontaining biological entities by probe hybridization. For example, themethods can be used to analyze cells, viruses, isolated nuclei orisolated chromosomes. The methods work by encapsulating biologicalentities in gel microdrops, such that at least some microdrops in apopulation contain a single entity. The gel droplet provides astabilization matrix for hybridization and holds hybridized nucleicacids together for subsequent analysis. Encapsulated entities arehybridized with one or more probes. GMDs are easily recovered using lowspeed centrifugation. Probes can, for example, be designed to hybridizeto particular chromosomes or to specific chromosomal loci which are thesite of genetic abnormality. After hybridization, the encapsulatedentities are detected and/or isolated based on hybridization signal.

The methods allow very large numbers of biological entities to beanalyzed simultaneously and can detect entities with rare genotypes fromwithin such populations. For example, the methods can be used toidentify rare cancerous cells in a population of normal cells at anearly stage in the development of the cancer. Other applicationsincluded gene identification, isolating cells expressing a particulargene, preparation of specific hybridization probes, and isolation ofpure starting material for DNA sequencing. An additional benefit is thatencapsulation in a permeable matrix permits hybridization in freesolution, improving the reaction kinetics. A further benefit is that theencapsulation matrix can serve as a repository for a substrate for areaction catalyzed by an enzyme bound to a probe. Use ofchemiluminescent substrates in this manner results in highly sensitivedetection.

II. Formation of Droplets

Gel Microdrop (GMD) encapsulation evolved from an interest in studyingindividual cells (1-11). GMD's provide a defined microenvironment arounda biologically entity. The gel does not impede diffusion and allowsanalysis of large numbers of individual GMDs using flow cytometry, aswell as recovery of GMDs of interest using FACS. The number ofbiological entities encapsulated within each GMD is approximated byPoisson statistics, similar to limiting dilution cloning or petri dishinoculation. To obtain a preparation with a high probability that eachGMD contains 0 or 1 initial chromosomes, about 10% of the GMDs should beoccupied. GMDs can be prepared by dispersing entities in liquefied gel,such as agarose, into an excess of a hydrophobic fluid to form anemulsion. The emulsion is transiently cooled, causing gelling. Onceformed, GMDs are physically distinct and robust and can be removed fromthe oil into an aqueous medium by low speed centrifugation.Alternatively, GMD's can be formed by passing a mixture of liquefied geland entities through a pulsating nozzle, such as the printhead of aninkjet printer.

Instrumentation for microdrop formation, the CellSys 100™ MicrodropMaker, is a specially designed emulsifier coupled to a high precisionmotor available from OneCell Systems, Inc. By varying the rotationspeed, type and amount of surfactant, and emulsion viscosity, microdropsranging from, for example, 2-200 μm can be prepared. Although theMicrodrop Maker currently available from One Cell Systems is mostefficient for making large numbers of microdrops (e.g., 10⁷), which inturn requires one million biological entities to meet the singleoccupancy requirement of the microencapsulation procedure, it can beminiaturized for encapsulation of smaller chromosomal preparations. Suchis useful for clinical applications, e.g., evaluation of bone marrowsamples in which only a small number of cells are present.

Several types of gel can be used for making droplets including agarose,alginate, carrageenan, or polyacrylamide. High melting temperatureagarose is preferred for encapsulating larger human and plantchromosomes.

III. Biological Entities

The methods are generally applicable for screening nucleic acids, andany biological entity containing them. Examples of biological entitiesinclude cells, organelles, such as nuclei, mitochondria andchloroplasts; chromosomes and fragments thereof, and viruses. Suchentities can be from any species including mammals, fish, amphibians,avians, insects, bacteria, eubacteria and plants. Preferred mammalsinclude humans, primates, bovines, and rodents, such as mice, rats andrabbits.

In some methods, biological entities are obtained from a tissue from ahuman patient. The tissue sample often contains a nonclonal populationof cells. Samples can be obtained from any tissue, but blood samples,and samples from tissues from the loci of diseases to which the patientis suspected of being susceptible are preferred. In some methods, cellsfrom primary tissue samples are propagated before analysis. In somemethods, biological entities are pooled from more than one individualbefore analysis. In some methods, biological entities (e.g.,chromosomes) are obtained from a homogenous cell line. Cells can beencapsulated and analyzed directly, or nuclei or chromosomes can beisolated from cells for analysis.

IV. Pretreatment of GMD's Before Hybridization

Preferably, polymers forming the gel are crosslinked to each otherand/or to the biological entity. Preferably, such crosslinking isreversible without damage to the biological entity, thereby allowing thebiological entity to be recovered after hybridization and subjected tofurther DNA manipulations. Such crosslinking assists in preservation ofstructural integrity of GMD's in the subsequent denaturation step andhybridization steps. Harsh chemical fixation treatments, such as theformaldehyde treatment, used in conventional FISH, are not required.Such fixation treatments form an internal matrix by cross-linkingendogenous primary amino groups in a biological entity.

Crosslinked gel microdrops can withstand high temperatures (at least68°) or concentrations of denaturing solvents such as formamide (e.g.,10, 20, 30, 40 or up to 50% concentration of formamide). Optionally,segments of DNA or RNA can be amplified within the droplets using PCR. APCR buffer including primers is diffused into droplets, and the dropletsare subject to temperature cycling as in a conventional PCR reaction.Irrespective whether amplification is performed, nucleic acids withinthe GMD's are typically denatured (e.g., by treatment with alkali, heat,formamide or other chemical denaturant) before performing thehybridization step.

V. Probes

Probes are designed to hybridize with selected segment(s) in the nucleicacids of biological entities being screened. Typical probes are thoseused in conventional genetic and cytogenetic analyses. In many methods,two or more probes with different binding specifies are used. In somemethods, a large population of different probes is used. Typically,probes bear detectable labels. If more than one type of probe is used,the different types sometimes bear different labels.

Some probes used in the methods are locus-specific probes, includingallele-specific probes and species-specific probes. Allele-specificprobes hybridize to one allele of a gene within a species withouthybridizing to other alleles. Similarly, species-specific probeshybridize to a gene from one species without hybridizing to the cognategene in another species. Some probes used in the method hybridize to avariant form of chromosome associated with disease without hybridizingto a wildtype form of the chromosome found in normal individuals. Someprobes are mixtures of probes designed to hybridize to one chromosomefrom an individual or species without hybridizing to other chromosomes.For example, a population of probes can be designed to hybridize to thehuman X chromosome without hybridizing to other human chromosomes. Someprobes are mixtures designed to hybridize to several differentchromosomes. For example, a mixture of probes can be designed tohybridize to each of the human chromosomes. Some probes hybridize tosatellite or repeat regions within chromosomes. Some probes hybridize tocentromeric regions of chromosomes.

Some probes are chromosome painting probes or reverse chromosomepainting probes. Chromosome painting probes are a collection of probesdesigned to hybridize to a segment of a chromosome. Microscopic analysisof a chromosome hybridized to such probes shows a contiguous segment oflabel if the entire segment of the chromosome is present. If the segmentis interrupted by a substitution, deletion or insertion, a gap appearsin the pattern of label, signifying the presence of a geneticabnormality. Reverse chromosome painting probes are designed tohybridize to a contiguous segment of a chromosome bearing a knownmutation. Microscopic analysis of a chromosome bearing such a mutationhybridized to reverse painting probes shows a contiguous segment oflabel.

Reverse chromosome painting has been useful for determining the originof de novo unbalanced chromosome duplications and the extent ofdeletions or balanced translocations (20). However, aberrant chromosomesare often difficult to distinguish in conventional FISH methods becausethe derivative chromosome can overlay normal chromosomes. Use of reversechromosome painting after separation of chromosomes by flow cytometryeliminates this problem.

Some probes are designed to bind to mRNA within a cell. Such probes canbe designed incorporating a segment from the antisense strand of a cDNAsequence.

Probes are typically nucleic acids, and can be RNA, DNA or PNA. Probescan also be antibodies or other proteins with capacity to bind to DNA ina sequence-specific manner.

VI. Labels

Probes are typically labelled. The labels used permit separation basedon flow cytometry and/or microscopic visualization of label. In somemethods, probes are labelled with fluorescent label such as fluorescein.If multiple probes are used simultaneously the probes can be labelledwith different fluorescent molecules emitting at different wavelengthsto allow differential detection.

In some method, the signal from a label attached to a probe is amplifiedby binding molecules bearing secondary labels to the label. For example,a hybridized probe labelled with fluorescein can be incubated 15-30 minwith rabbit anti-fluorescein IgG conjugated with biotin (AccurateChemical & Scientific). After washing with PBS buffer, GMDs areincubated for 15-30 min with avidin-FITC or avidin-phycoerythrin (Sigma,St. Louis, Mo.). Because, on average, each anti-fluorescein is labeledwith five biotin molecules and each biotin molecule can bind 2-4 avidinmolecules, a 10-20 fold amplification in signal is obtained.

In some methods, probes are labelled with an enzyme that catalyzesconversion of a substrate to a secondary label that allows separationand/or visualization of GMD's.

In some methods, as well as being hybridized to sequence-specificprobes, GMD's are labelled with compounds that binds to any DNAsequence. Such labelling serves to distinguish GMD's containing abiological entity with empty GMD's.

VII. Separation and Analysis of Hybridized GMDs

After hybridization with labelled probes, GMDs can be analyzed on a flowcytometer. In the simplest case, in which a single probe type is used,the flow cytometer counts the number of GMDs bearing a label and thenumber of GMD's lacking a label. If two different probes bearingdifferent labels are used, the flow cytometer can count GMD's bearingfirst label only, GMD's bearing second label only, GMD's bearing bothlabels, and GMD's bearing neither label. In methods employing largernumbers of probes, still further categories of GMD's can bedistinguished.

If a probe is directed to a particular sequence (e.g., a specificchromosomal defect), detection of GMD's hybridized to that probe signalsthat the defect is present in at least some of the biological entitiesbeing analyzed. If all GMD's bearing biological entities are labelledwith a second label, it is possible to determine a ratio of the GMD'shybridized to the specific probe with all GMD's encapsulating abiological entity. This ratio is the proportion of biological entitiesin a sample preparation bearing a defect. The methods are sufficientlysensitive to detect rare cells in larger populations, e.g., one cell in10, 100, 1000, 10,000, 100,000 or 1,000,000. This ratio can besignificant in determining the existence or prognosis of a disease. Forexample, if a sample of a cell is from a tissue suspected of beingsusceptible to cancer, the ratio of cells bearing a defect associatedwith cancer to the total number of cells in a sample is a measure of howfar the cancer has progressed.

In some methods, GMD's encapsulating chromosomes are hybridized with twodifferent probes which are complementary to segments normally found ondifferent chromosomes but which are translocated into the samechromosome in cancerous cells. In this situation, the ratio of GMD'sbinding to both probes relative to the GMD's binding to one probe or theother but not both, gives the ratio of cancerous to normal cells in apopulation.

Optionally, flow cytometry can be followed by FACS sorting to makedifferent classes of GMD's available for further analysis, such asmicroscopy, or chromosome preparation. Alternatively, gel microdropletscan be labelled with magnetic particles and subjected to magneticseparation (MACS). Magnetic particles can be directly attached tohybridization probes or can be supplied in a form that they specificallybind to hybridization probes.

VIII. Visualization of Hybridized Chromosomes

Encapsulated biological entities can be visualized with or without priorflow cytometry and FACS separation to determine the location(s) at whichprobe has bound. Analysis after flow cytometry and FACS separation canbe advantageous because at that stage one has a relatively purepopulation of biological entities that has hybridized to a given probe.Biological entities can be visualized by microscopy, digital imageanalyzing, scanning cytometry, photon counting or ccd. Visualization isuseful for analyzing hybridization of chromosome painting probes orreverse chromosome painting probes. Visualization is also useful fordetermining chromosomal copy number within a cell, and hence theexistence of chromosomal deletions or duplications. For biologicalentities containing multiple chromosomes (e.g., cells and nucleic)visualization can also be used to distinguish between two differentprobes binding to separate chromosomes or to the same chromosome. Asnoted, such analysis is useful in identifying some forms of cancer.

Biological entities are preferably immobilized on microscope slides orthe like for visualization. For example, placing a small quantity (10μl) of GMDs dispersed in substrate on the glass slide with a cover slipsufficiently immobilizes GMDs permitting reliable detection of emittedlight.

Digital imaging has become an indispensable tool for biological researchdue to several advantages when compared to the human eye. The highersensitivity imaging detector enables one to visualize very low lightobjects which are not detectable by the unaided human eye. The spectrumsensitivity of the human eye is limited from 400 to 700 nm. In contrast,the spectrum sensitivity range of imaging detectors is more broad, andsignals from the range of x-ray to infrared can be detected.

A charged-coupled device (CCD) camera providing exposures ranging fromseconds to minutes and has advantages for detecting low light levels.These cameras are coupled to a microscope; then digital images arecollected with the help of appropriate instrumentation. For low lightapplications, there are two types of CCD cameras available. The first isthe Intensified CCD (ICCD) camera which uses an Image Intensifier and aCCD camera. The Image Intensifier enhances low light image and theintensified image is projected onto a CCD camera through relay optics,such as a relay lens, enabling one to visualize low light imageundetectable using a CCD alone. The second is a Cooled CCD (CCCD) camerawhich uses a similar CCD chip for high light imaging. The CCCD reducescamera noise by cooling and slowly reading out the signal. The reductionof noise enables one to visualize a low light image ordinarily buried inthe noise of a regular CCD camera.

IX. DNA Isolation

The gel environment preserves chromosomal DNA molecules in intact form.DNA in encapsulated chromosomes containing GMD's can be cleaved tofragments in situ with restriction enzymes. For large fragments, partialdigestion is preferred. DNA fragments can then be released from drops bydigesting the gel matrix. For example, if the matrix is agarose, the gelmatrix can be digested with the enzyme agarose. Large fragments are thencloned into vectors such as YACs, BACs or PACs. Libraries from purifiedhuman chromosomes preparable by the above methods are useful forsequencing or mapping the human genome and for positional cloning. Puresorted chromosomes are also useful as a source of chromosome paintingand reverse chromosome painting probes. Such probes can be prepared byamplification of chromosomal DNA using degenerate primers.

X. Storage of Encapsulated Biological Entities

Encapsulated biological entities such as chromosomes can be stored foran hour, a day, a week, a month, six months, a year, two years or fiveyears or more without visible degradation of nucleic acids. Storedchromosomes can eventually be used for isolating specific genes, forpreparing PCR probes, and for generating high quality starting materialfor DNA sequencing, or for clinical analysis.

XI. Applications

The above methods can be applied to diagnosing the presence,susceptibility, or prognosis of diseases associated with geneticdefects. The methods are particularly useful in diagnosing andmonitoring diseases due to genetic defects that are only present in asubpopulation of cells, such as defects arising from somatic mutation.Examples of such diseases associated with genetic defects includeautoimmune diseases, inflammation, cancer, diseases of the nervoussystem, and hypertension. Some examples of autoimmune diseases includerheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependentand non-independent), systemic lupus erythematosus and Graves disease.Some examples of cancers include cancers of the bladder, brain, breast,colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary,pancreas, prostate, skin, stomach and uterus.

Many cancers arise due to mutations in rare subpopulations of cells.Cancers develop and progress through the accumulation of geneticabnormalities at critical loci (34, 35). These abnormalities may involvealterations of one or a few bases of DNA, deletions ranging fromsub-microscopic to whole chromosomes, duplications or higher-levelamplifications of chromosomal regions, or rearrangements producingabnormal juxtapositions of DNA sequences (36). In some case, such as theabl-bcr fusion on the original Philadelphia chromosome, specifictranslocations are associated with activation or modification of humanproto-oncogenes (37, 38). Similarly, Ewing's sarcoma is associated witha translocation involving the EWS gene on chromosome 22. Determinationof the proportion of cancerous cells in a tumor allows grading of thetumor for improved diagnosis, prognosis and treatment planning. Theratio can also be valuable in evaluating both the adequacy of surgicalmargins and the presence of microscopic metastases in bone marrow orother sites.

The methods are also useful for diagnosing the presence of latentviruses. For example, some viruses, such as Herpes viruses andretroviruses, integrate into genomic DNA of some cells within the bodyand remain dormant until activated. Analysis of a tissue sample from apatient having or suspected of being infected with such a virus canidentify the percentage of cells infected with the virus and the copynumber of the virus in different cells. Such information is useful indiagnosing the presence of virus, the severity of infection and therecommended course of treatment. Presence of a virus can be detectedusing a probe designed to hybridize either to viral genomic nucleic acidor to viral mRNA, or both. The methods can similarly be used todetermine copy number of viral mRNA in cells from the patient. Suchinformation can be useful in monitoring the progress of disease, forexample, in response to treatment with a drug.

The methods are also useful for determining allelic frequencies in apopulation, and correlating such frequencies with a phenotype. Forexample, cells taken from a population of individuals can be pooled, andscreened to determine the frequencies of different allelic forms of agene. If the population has a common phenotype (e.g., a disease), acorrelation can be performed to determine whether the presence of one ofthe allelic forms is statistically associated with the phenotype.

The methods are also useful for identifying cell types expressing a geneof interest. For example, if a new gene of unknown function has beendiscovered, one can design a probe that is complementary to an exonicsegment and optionally, to segments in successive exons. Hence, theprobe can hybridize to mRNA expressed from the gene. A population ofcells is obtained from different tissues of an individual, and the cellsare screened for hybridization to the probe. Cells hybridizing to theprobe express the gene. The nature of cells expressing a gene providesvaluable information concerning the function of the gene.

Similar methods can be used to clone a cDNA if only a portion of thecoding sequence is known. The portion of known sequence is used todesign a probe. A population of different cells is then encapsulated andhybridized with the probe. Cells are then separated according to extentof expression. Cells showing the highest level of expression provide asuitable source material from which to clone the cDNA.

The methods are also useful for comparing or monitoring the expressionof a given gene or gene(s) in different cell types. A probe is designedto hybridize to a mRNA transcript of each gene of interest. Optionally,different probes can bear different labels. Probes are then hybridizedwith mRNA in a microdrop encapsulated cell population, which typicallyincludes cells of different types. The extent of hybridization of eachcell with each probe is then determined. Optionally, cells hybridizingwith a particular probe at significantly above or below average levelsare isolated and cell type determined, allowing correlation between celltype and expression level. Optionally, different cell types in apopulation can themselves be labelled with reagents that specificallybind to a particular cell type. For example, a particular cell type canbe labelled using an antibody that binds to a receptor specific to thecell type. Microdrops are then analyzed for both cell type labels andprobe labels, thereby facilitating comparison of expression levels ofparticular mRNA species between the cell types that have beenspecifically labelled.

The methods are also useful for preparing isolated chromosomes. Asnoted, isolated chromosomes are useful for e.g., positional cloningstudies and for preparing probes.

XII. Kits

The invention also includes kits for the practice of the methods of theinvention. The kits comprise equipment and/or reagent(s) for making gelmicrodrops and optionally, probe(s) for performing hybridization toencapsulated nucleic acids. Examples of equipment include a CellSys 100™Microdrop Maker and components thereof, an instrument providing apulsating novel, such as an inkjet printer, and a vortexer. Examples ofreagents include chemicals for making a gel, such as agarose oracrylamide, cross-linking reagents, denaturing agents, and hybridizationbuffer. The kits can also include label(s) and other chemicals toamplify label signal. The kits usually include labelling or instructionsindicating the suitability of the kits for performing hybridization ingel drops and/or flow cytometrix analysis. The term “label” is usedgenerically to encompass any written or recorded material that isattached to, or otherwise accompanies the kit at any time during itsmanufacture, transport, sale or use.

EXAMPLES Example 1 Encapsulation, Hybridization and Screening ofChromosomes, and Stability of Encapsulated Chromosomes Materials andMethods Cell Lines and Culture Conditions

Human chronic myelogenous leukemia, K-562, human acute lymphocyticleukemia, REH (American Type Culture Collection, Rockville, Md.), andmouse fibroblast, Mus Spretus C1-5A (Los Alamos National Laboratory, LosAlamos, N. Mex.) cells were grown in RPMI 1640 medium supplemented with10% fetal bovine serum at 37° C. in the presence of 5% CO₂. Normal humanlymphoblast cells GM130 (NIGMS Human Genetic Mutant Cell Repository,Coriell Institute for Medical Research, Camden, N.J.) were grown underidentical conditions, except that RPMI 1640 medium was supplemented with15% heat inactivated fetal bovine serum.

Source of Plant Chromosomes

Plant chromosomes from the field bean Vicia faba, (from J. Dolezel,Institute of Experimental Botany, Olomouc, Czech Republic), wereisolated from root meristems after cell cycle synchronization withhydroxyurea and metaphase accumulation with amiprophos methyl (21).

Mitotic Cell Preparation

Mouse C1-5A cells (an adherent cell line) undergoing logarithmic growthwere treated with 0.2 μg/ml colcemid (Sigma, St. Louis, Mo.) for 12-15hours. Mitotic cells, which become less adherent, were shaken-off andresuspended in hypotonic solution (55 mM KCl). Human K-562 cellsundergoing exponential growth were treated with 60

M genistein (Sigma) for 24 hours to synchronize growth (23). Cells werethen pelleted and washed once with Hank's balanced salt solution(Sigma). Fresh media containing 0.1 μg/ml of colcemid was added andcells were grown for an additional 24 hours. After counting, cells werepelleted by centrifugation (100 g for 10 min at 4° C.) and resuspendedin hypotonic solution (75 mM KCl). Human REH cells were grown tostationary phase, to synchronize growth, then were left for 2 days.Cells were harvested by low speed centrifugation and grown in freshmedia containing 0.1 μg/ml colcemid for 24 hours. After pelleting, cellswere resuspended in hypotonic solution (75 mM KCl).

Chromosome Isolation

Human and mouse chromosomes were isolated using the polyamine method(31,32), with minor modifications. Approximately 2×10⁷ cells wereswelled in 10 ml of hypotonic solution either for 30 min (human REH) orfor 1 hr (human K-562 and mouse C1-5A cells) at room temperature. Afterswelling, cells were pelleted by 5 min centrifugation at 40 g, andgently resuspended in 0.1 ml of fresh hypotonic solution. Two ml ofchromosome isolation buffer (CIB; 15 mM Tris-HCl, 80 mM KCl, 20 mM NaCl,2 mM EDTA, 0.5 mM EGTA, 0.2 mM spermine, 0.5 mM spermidine, pH 7.2)supplemented with 0.1% (v/v) 2-mercaptoethanol and 0.2% (v/v) TritonX-100 was added and solutions were mixed by brief (10 sec) vortexing.Tubes were kept at 0° C. for approximately 10 min. C1-5A cells werepassed through a 25 gauge needle until chromosomes were released, asmonitored by fluorescence microscopy. Nuclei and cellular fragments wereremoved by three successive 5 min centrifugations at 40 g. The top twothirds of the supernatant, after the final centrifugation, was kept at4° C. for 16 hr. The supernatant was then carefully decanted to avoiddisturbing the settled chromosomal pellet. Chromosomes were resuspendedin 0.2 ml of CIB and aggregates were pelleted by centrifugation for 5min at 40 g. Chromosomes were either stored in CIB or immediatelyencapsulated in agarose gel microdrops (GMDs).

Chromosome Encapsulation

The chromosome/agarose mixture (2.3% Type XII agarose; (Sigma) 0.1%Triton X-100 (Sigma) containing 10⁶ chromosomes/0.55 ml) was prepared bymelting the agarose in CIB at 100° C., cooling the agarose to 58° C.,and adding 100 μl of chromosome suspension and 50 μl of 11% (v/v) TritonX-100 (pre-warmed to 58° C.) to 0.4 ml of melted agarose. The mixturewas held at 58° C. for 5 min and added dropwise to 15 ml of CelMix™ 200emulsion matrix (One Cell Systems, Cambridge, Mass.) which was alsopre-warmed to 58° C. GMDs were generated with a CellSys100™ MicrodropMaker (One Cell Systems, Cambridge, Mass.) equipped with a 1.6 cm bladeusing successive rotor speeds of 1,500 rpm for 1 min at 20-25° C., 1,500rpm for 1 min at 0° C., and 1,500 rpm for 5 min at 0° C. GMDs wereseparated from the emulsion matrix by centrifugation at 350 g for 10min. The encapsulated chromosome-containing pellet was washed twice with13 ml of CIB, re-pelleted by centrifuging 5 min at 250 g, and stored at4° C. in 10 ml of the same buffer.

Chromosome Staining

Encapsulated chromosomes were stained with propidium iodide (0.4 μg/ml)for microscopic examinations and with 7-actinomycin (7AAD) at the sameconcentration, both from Sigma, for flow cytometry.

Hybridization Probes

LSI™ 22q (bcr-locus specific, Vysis, Downers Grove, Ill.), whichhybridizes to a 300 kb region of the bcr gene on chromosome 22, wasused. This probe was directly labeled with Spectrum Green (fluorescein).After labeling, the probe size distribution ranged from 50-500 nt. TheDNA probe was denatured at 95° C. for 5 min before hybridization.

Microdrop In Situ Hybridization (MISH) of Human Chromosomes

Encapsulated chromosomal DNA was denatured in 0.1 N NaOH, 50% (v/v)ethanol for 1.5 min. GMDs were washed once in 0.5 M sodium carbonate, pH10.2. After denaturation, the hydroxyl groups in the agarose microdropswere mildly crosslinked with 5

M divinylsulfone by incubating 30 min at room temperature. Excessreactive groups from divinylsulfone were blocked with 2% (v/v)2-mercaptoethanol for 30 min in 40 mM Tris-HCl, pH 8.0. GMDs were thenwashed with 2×SSC (0.15M NaCl, 0.015M sodium citrate).

100

(approximately 2×10⁵) of human chromosomes encapsulated in GMDs washybridized with 2.0 μl of the denatured probes (probe concentration wasproprietary for the manufacturer) in a hybridization mixture (2×SSC, 10%(v/v) dextran sulfate, 50 μg/ml salmon sperm ssDNA for 16 hours at 68°C. After hybridization, non-specifically bound probe was removed byincubating GMDs with 1.0 ml 0.4×SSC at 72° C. for 5 min (LSI/22) andwashing twice with 0.4×SSC at room temperature. Cot DNA was added toprevent non-specific hybridization to repetitive sequences.

Microscopy and Digital Image Analysis

The integrity of chromosomes after fluorescent staining or MISH waschecked visually using an Olympus BH-2 microscope (40× SPlan 0.4objective or phase contrast A40 PL 0.65 objective) equipped forepifluorescence with appropriate filters for fluorescein and 7AAD.

Flow Cytometric Analysis

After staining or MISH, encapsulated chromosomes were analyzed using aFACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems,San Jose, Calif.) equipped with a standard 100 μm nozzle. LYSYS IIVer.2.0 software was used for data analysis. To remove large particles,the GMDs were sieved through a 53 μm nylon mesh (Small Parts Inc., MiamiLakes, Fla.). For flow cytometry analysis, a concentration of microdropsnot exceeding 2×10⁵/ml was used. Forward and side scatter signals wereanalyzed on a log scale and examined in scatter plot format, permittingidentification of and gating on GMDs. 7ADD fluorescence was used toidentify GMDs containing encapsulated chromosomes. To identify 7ADD, anargon laser with a 356 nm spectral line was used. To identifychromosomes which hybridized to the Spectrum Green™ labeled probe,fluorescein (FITC) intensity was measured. For measuring FITCfluorescence, an argon laser with a 488 nm spectral line was used.

Fluorescence Activated GMD Sorting

Encapsulated chromosomes were sorted using a FACS Vantage fluorescenceactivated cell sorter (Becton Dickinson) adapted with a macrosortoption. The sheath pressure was set at 2 psi with a sample differentialof 1 psi. A large diameter sample line was used to avoid clogging. TheGMD samples were sieved through a 53

m mesh before sorting. Sorting speed was in the range of 50 FITC-labeledchromosomes/sec. Sorted GMD-encapsulated chromosomes were pelleted bylow speed centrifugation and taken up with 20

l of Antifade solution (Oncor, Gaithersburg, Md.), diluted 1:1 with CIBand analyzed using a fluorescence microscope.

Digestion of Denatured Encapsulated Chromosomal DNA with HindIII

Encapsulated chromosomes were denatured, as previously described, washedwith 40 mM Tris-HCl, pH 8.0 and subsequently digested with Proteinase K(2 mg/ml) supplemented with lithium dodecyl sulfate (1%) in CIB for 12hours at 50° C. Chromosomes were then washed three times with a 200-foldexcess of CIB and two times with 20-fold excess of HindIII digestionbuffer (50 mM NaCl/10 mM Tris-HCl, pH 8.0/10 mM MgCl₂). Digestion ofencapsulated chromosomes was done using 20 units of HindIII (Gibco-BRL)in a reaction volume of 50 μl for 90 min at 37° C. Gel loading bufferwas added and samples were electrophoresed as described below.

Gel Electrophoresis of Chromosomal DNA

Both free and encapsulated chromosomes were treated with proteinase Kand lithium dodecyl sulfate as described above for three hours at 50° C.Gel-loading buffer (6×, 0.25% bromphenol blue, 0.25% xylene cyanol FF,30% glycerol) was added and samples were electrophoresed on 0.8% SeaKemGold (FMC BioProducts, Rockland, Me.) agarose in TAE buffer (0.04 MTris-acetate, 1 mM EDTA) at 56 volts for 2-6 hours. Propidium iodide waspresent in the gel (0.5 μg/ml) and in the electrophoresis buffer (0.05

g/ml) during electrophoresis. 0.25 μg of lambda DNA or 1.0 φgöX174 DNAcut with HinFI (Gibco-BRL) was used as a standard.

Chromosome Storage and Recovery

Free and encapsulated chromosomes were kept at 4° C. for up to 6 monthsin CIB. Agarose (from Pseudomonas atlantica, Sigma) was used to digestGMDs to recover chromosomes of interest. Encapsulated chromosomes werepelleted, resuspended in phosphate buffered saline solution, pH 7.0(Sigma), and agarose (30 units per 1000 GMDs) was added. This suspensionwas incubated at 40° C. for two hours, treated with proteinase K asdescribed above, and chromosomal DNA was analyzed by gelelectrophoresis.

Results

FIG. 1 shows encapsulated and unencapsulated human, mouse, and plantchromosomes. Encapsulated chromosomes appear visually more compact thanunencapsulated chromosomes, with narrow centromeric regions and tightlybound chromatid extensions. The centromeric region of encapsulatedchromosomes appear physically unseparated, which would be an indicationof chromatid loss. Interestingly, encapsulation of intact plantchromosomes, which are approximately 3 times larger than humanchromosomes, was also successfully performed using this procedure.

Stability of Encapsulated Chromosomes

To assess the long term stability of encapsulated chromosomes, weexamined DNA fragmentation using gel electrophoresis. As depicted inFIG. 2, no DNA fragmentation was found by electrophoretic analysis, even6 months after encapsulation. In contrast, one month after isolation,DNA from unencapsulated human and mouse chromosomes has a smearedbanding pattern, indicative of DNA fragmentation due to nucleasecleavage. This result indicates that encapsulated chromosomal DNAremains intact and can be used for preparing chromosome-specificlibraries. Intact isolation and long term stability of high quality,high molecular weight DNA will be a major convenience for researchersand an innovation for sample storage for clinical use.

In Situ Hybridization of Encapsulated Chromosomes

An important improvement was development of a matrix crosslinking methodwhich allowed use of up to 50% formamide and temperatures as high as 95°C., necessary for reproducible hybridizations. The agarose hydroxylgroups were mildly crosslinked with divinyl sulfone. Although thisprocedure also partially crosslinks chromosomal DNA to the agarosematrix, because this process is reversible at pH 10, DNA can be releasedfrom GMDs after MISH, which is important for eventual construction ofchromosome-specific libraries.

Using a FACS Vantage both for flow cytometric analysis and cell sorting,dual-parameter dotplots were produced by plotting FITC-fluorescence,which was detecting probes hybridized to the chromosomes versus 7AADfluorescence, which as a general DNA stain was used to detect allchromosomes.

FIG. 3A shows flow sorting of human chromosome 22 following microdropsin situ hybridization using a bcr locus specific probe. Chromosomes werecounter-stained with 7-amino-actino-mycin (Vysis, Downer's Grove, Ill.).Chromosomes were counter-stained with 7AAD. R1 includes high FITC, low7AAD fluorescence specific for gel microdrops containing chromosome 22.The scattergram also display empty GMDs, encapsulated chromosomes, andnoise. FIG. 3B shows sorted chromosomes visualized using fluorescentmicroscopy. The hybridization signal is depicted in green (FITC) color.Hybridization signals were amplified using the TSA method, whichprovides a 10-100 increase in signal intensity.

FIG. 4 shows purity of chromosome 22 after sorting measure by thepolymerase chain reaction using primer sets specific from chromosomes10, 21 or 22. Amplification results for each primer set were comparedusing unsorted and sorted chromosome populations. Unsorted chromosomesgenerated specific amplicons for each primer (lanes 3-5), Sortedchromosome 22 showed no contamination with chromosome 10 (lane 8 vs. 60and were less than 1% contaminated with chromosome 21 (lane 8 vs. lane7).

Recovery of Encapsulated Chromosomes

We determined that after recovering chromosomes from agarose microdrops,DNA was not fragmented, even 6 months after encapsulation. We alsotested the integrity of DNA after digesting microdrops with agarase andfound that the electrophoretic pattern was the same as that of untreatedencapsulated chromosomes used as controls. These observations show thatafter release from gel microdrops, encapsulated chromosomes will be aconvenient source of high quality DNA for molecular genetics studies.

Digestion of Denatured Encapsulated Chromosomes

A major concern for eventual use of sorted chromosomes to constructchromosome-specific libraries was that digestion of chromosomal DNA withrestriction enzymes, which is necessary for cloning large DNA fragments(>100 kb) into BAC libraries, would be impossible because denaturationwould destroy the DNA secondary structure. To address this concern, wetested the hypothesis that brief alkaline use would only partiallydenature the DNA and that subsequent digestion with proteinase K at 50°C. in low salt buffer overnight would restore double strandedness, thusmaking specific enzyme digestion possible. Human chromosomes isolatedfrom the GM 130 cell line were digested with proteinase K and cleavedwith HindIII. The electrophoretic result depicted in FIG. 5 shows thatthe restriction endonuclease HindIII cleaved restored DNA and generatedtypical sized fragments, demonstrating that MISH treated chromosomes canbe used for library construction.

Example 2 Construction of Chromosome-Specific BAC Libraries

A major unrealized goal of fluorescence in situ hybridization assays hasbeen the use of flow cytometry to isolate specific chromosomes forlibrary construction. Prior to development of the MISH method, afterhybridization, only a small fraction of chromosomes remain intact andfree in suspension. Without the protection gained using the agarosemicrospheres, most chromosomes are clumped or fragmented, making themlargely unsuitable for flow cytometric analysis (27). We have shown notonly that we can hybridize and flow sort encapsulated chromosomes, butalso that alkaline-denatured chromosomes can be digested withrestriction endonucleases. As a result of this finding, we proposeconstruction of chromosome-specific libraries. Human chromosome 21 waschosen because it is often difficult to identify and sort usingconventional dual fluorescent staining since it is small andindistinguishable in the presence of cellular debris (29). The secondBAC library will be constructed using human chromosomes 9, which belongsto the group of chromosomes not resolvable by conventional dualfluorescent staining because of its similarity in size to several otherchromosomes (13).

Chromosome libraries are constructed using the steps below:

Example 3 Use of MISH-Sorted Chromosomes for Reverse Chromosome Painting

The development of in situ hybridizations with flow sorted chromosomelibraries (25,26), combined with non-isotopic signal detection (30), hasbecome a powerful approach for rapidly analyzing human chromosomalaberrations, such as aneuploidy and translocations. These techniques,termed chromosome painting, are becoming widely used in clinicalcytogenetics. However, small rearrangements, additions, or deletions arenot detectable using conventional chromosome painting, but theseaberrations are detectable by reverse chromosome painting, which isperformed using a probe prepared from aberrant chromosomes.

A method of using MISH-sorted chromosomes is depicted below:

Example 4 Detection of the Philadelphia Chromosome

The Philadelphia chromosome is a shortened chromosome 22 that resultsfrom a balanced translocation between chromosomes 9 and 22 with thetranslocation breakpoints at 9q34 and 22q11. As a result of thistranslocation, most of the abl oncogene, located on chromosome 9, isjuxtaposed to part of the bcr gene, located on chromosome 22, creating anew bcr-abl gene fusion (40, 51, 52, 53, 54). This gene fusion encodesan abnormal protein with strong tyrosine kinase activity compared withthe weak tyrosine kinase activity of the normal abl protein. Theabnormal tyrosine kinase produced from bcr-abl causes increased cellproliferation and contributes to leukemogenesis by unknown cellularpathways.

Translocations such as bcr/abl are currently detected by cytogeneticexamination of metaphase chromosome preparations prepared from bonemarrow cultures. Although this method is adequate to detect most chronicmyelogenous leukemia cases (CML) in which Ph¹-positive chromosomaltranslocations are visible after banding due to the size difference inmetaphase spreads, in acute lymphoblastic leukemia (ALL), cytogeneticexamination is successful in only 65-80% of the cases, depending on theexperience of each laboratory (55). The lower yield in ALL is the resultof the following factors. First, many ALL patients have inaspirable bonemarrows, so no cells are available. Second, lymphoblasts can bedifficult to culture, so there is little enrichment of leukemia cells.And third, the percentage of Ph¹ carrying cells in a sample may be aslow as 30%, in contrast with CML in which virtually 100% of thecancerous cells in the sample carry Ph¹. In cases where chromosomalrearrangements are not visible in classical metaphase spreads, which canoccur in Ph¹-positive chromosomes, both in CML or ALL leukemias,fluorescence in situ hybridization, Southern blot (DNA) analysis, orpolymerase chain reaction (PCR) analysis must be performed to detectrearrangements (55).

In CML, Southern blot analysis has been useful in detecting evidence ofPh¹ chromosome in patients in whom the cytogenetics are negative despitea clinical presentation of CML (53). This approach is only of limitedutility in ALL because only 25-50% of Ph¹ chromosome positive ALLpatients have M-bcr (Major breakpoint rearrangements). Nearly all M-bcrnegative patients have translocation breakpoints in m-bcr (minorbreakpoint rearrangements) anywhere in the first intron, which is 70-kbin size, requiring performance of an impractical number of Southernblots in order to adequately investigate this entire region.

The chromosomal breakpoints in CML and acute leukemias may occur overlarge DNA sequences: 5.8 kb for the bcr region in CML and over 90 kb forthe first intron of the bcr 1 gene in acute leukemias. Therefore, PCRamplification of the fusion bcr/abl gene sequences cannot be performedon DNA from patient specimens. All PCR based methods are designed toamplify and detect the abnormal fusion of mRNA (RT PCR). The design ofthese methods takes advantage of the fact that mRNA sequences are muchshorter, lacking intron sequences. Assuming primers can be designed toamplify short stretches of mRNA specific for particular translocation(currently available for Ph¹ fused mRNA), amplified target can bedetected after hybridization with specific probes followed by gelelectrophoresis and Northern blotting. The size of bcr/abl fused mRNAfor different patients varies within certain ranges for CML and acuteleukemia breakpoints, but the same size fusion mRNA is characteristicfor each malignant clone and can be monitored for each patient over theclinical course of the leukemia.

An advantage of using mRNA as a target for PCR amplification is thatmitotic cells are unnecessary. The practical limit of sensitivity ofdetection is approximately 1 malignant per 10,000 non-malignant cells.While the exquisite sensitivity of PCR could be advantageous inmolecular diagnostic testing, its use is troublesome in the clinicallaboratory. Contamination with minute amounts of amplified DNA and/orRNA from patient samples or cell line controls, even in the range of 1to 10 copies, may generate false-positive results. Aerosols andcarryover are the main sources of contamination. A potential problemwith monitoring the presence of the Ph¹ chromosome after chemotherapy orbone marrow transplant by RT PCR is the presence of residual deadleukemia cells with intact mRNA complicating therapeutic assessment.

The MISH technique combined with flow cytometric analysis makes asignificant contribution to analysis of translocations. Becausemetaphase chromosomes are required, only live leukemia cells contributePh¹ chromosomes for detection. As long as leukemia cells proliferate andreach mitosis, approximately 20 Ph¹ positive chromosomes in the presenceof 50,000 chromosomes, including those from Philadelphia-negative cells,can be detected in about 5 min. This corresponds to detection of 20cells with a single copy of Ph¹ chromosome, or 10 cells with twodefective copies, in an environment of approximately 1,000 cells whichare Ph¹ chromosome negative (2% leukemia cells present). To improvestatistical significance, analysis of 100,000 events can be performed inapproximately 10 minutes, provided that metaphase chromosomes areavailable from both cell types. In contrast, in current FISH procedureusing metaphase spreads obtained from bone marrow cultures which have amitotic index of 10%, one would have to find one Ph-positive spread inthe presence of 1,000 Ph-negative spreads, a labor intensive andimpractical approach by conventional microscopy.

Materials and Methods

Chromosome encapsulation and hybridization conditions were as describedabove. The LSI™ bcr/abl DNA probe was used for in situ hybridization.The probe was directly-labeled with SpectrumGreen™/SpectrumOrange™ whichwas designed to detect translocations between chromosome 9 and 22(Vysis, Downers Grove, Ill.). This probe detects bcr/abl gene fusions,the molecular equivalent of the Philadelphia chromosome (Ph¹), in bothmetaphase and interphase cells. It can be used to identify bcr/abl genefusion involving either of the two breakpoint regions (M-bcr and m-bcr)in the bcr gene on chromosome 22. The bcr/abl translocation probe isqualified for use on both cultured lymphocytes and bone marrow cells.The LSI™ probe does not contain repetitive sequences and is composed ofan abl probe directly labeled with SpectrumOrange fluorophore and a bcrprobe directly labeled with SpectrumGreen fluorophore. The abl probebegins between c-abl exons 4 and 5 and continues for about 200 kb towardthe telomere of chromosome 9. The bcr probe begins either between bcrexons 13 and 14 (m-bcr) or between bcr exons 2 and 3 (M-bcr) and extendstoward the centromere approximately 300 kb crossing well beyond them-bcr region. The probe size distribution is within a range of 50-500 nt(after labeling).

After hybridization, chromosomes containing M-bcr/abl gene fusion inChronic Myelogenous Leukemia (CML) and in Acute Lymphoblastic Leukemia(ALL) can be expected to contain fused orange and green signals intranslocated chromosome 22, which are sometimes perceived as yellow.Normal chromosomes 22 should display green signal and normal chromosomes9 should display orange signal. Hybridized chromosomes that have them-bcr/abl gene fusion in ALL should contain fused green/orange signal inchromosome 22 and faint green signal not fused with orange signal onchromosome 9 (from the chromosome 22 region between m-bcr and M-bcr thatis translocated to chromosome 9).

Results Chromosome Isolation

To increase the yield of human mitotic cells, K-562 cells weresynchronized by a novel method using genistein, an isoflavone whichblocks the cell cycle at G₂/M. An important advantage of genistein forcell cycle synchronization was that it did not appear to penetrate cellsand was easily eliminated by washing. After synchronization, growth ofK-562 cells was not significantly affected by the presence of colcemidfor up to 24 hours, and a high percentage (75%) of cells, therefore,reached mitosis. Use of genistein yielded a ready supply of millions ofchromosomes facilitating extensive experimentation with encapsulationand fluorescent in situ hybridization conditions. The method can also beused to isolate chromosomes for other purposes.

In FIG. 6, detection of translocated chromosome 22 left chromosome 9middle, and translocation-free chromosome 22 right by MISH are shown.Translocated chromosome 22 is identifiable by the presence of both greenand red colors on a background of a blue colored whole chromosome. Fusedred and green signals may be perceived as yellow (bottom left of FIG.6). This yellow color also represents fused bcr/abl gene (translocatedchromosome 22). Translocation-free chromosome 22 is detectable by thepresence of single green color and chromosome 9 by the presence ofsingle red color. Digital images created by a CELLscan digital imagesystem with Exhaustive Photon Reassignment (EPR), which was availablethrough collaboration, are presented here, but a fluorescence microscopeequipped with a triple bandpass filter for DAPI, fluorescein andrhodamine was adequate to identify translocations in Philadelphia¹chromosome.

Example 6 Analyzing Encapsulated Human Nuclei Using Microscopy and FlowCytometry

Analyzing MISH signals in nuclei provides improved contrast relative toMISH detection in chromosomes. Moreover, MISH analysis of nuclei can beperformed in cases where cell proliferation is difficult or impossible.The applications are numerous and include detection of: translocations,deletions, monosomies or trisomies for diagnostic and prognosticanalysis of aberrations. This method can also be used in researchapplications including gene amplifications and gene mapping. In thisexample, we use human nuclei with deleted chromosome 3 as a modelsystem. This genetic defect presents numerous clinical symptoms inaffected people including mental retardation and multiple congenitalanomalies.

Isolation of Nuclei

Nuclei are isolated from human lymphoblastoid cell line GM11428 (NIGMSHuman Genetic Mutant Cell Repository, NIH, Bethesda, Md.). HL-60 cellsare a source of normal nuclei (control) with no deletions in anychromosome. Cells in logarithmic phase of growth are collected andswelled in hypotonic solution (75 mM KCl) for 1 hour. Cells are thenpelleted by low speed centrifugation and resuspended in2-mercaptoethanol-free CIB buffer (1 million cells/ml). Triton X-100 isthen added to obtain final concentration of 0.2%. Nuclei released fromcells are kept in this solution for 1 hour at 4° C., then pelleted bycentrifugation at 100×g and resuspended in original cell volume of thesame buffer and re pelleted. Finally, nuclei are resuspended at aconcentration of 10 million nuclei per ml of 2-mercaptoethanol-free CIBbuffer.

Encapsulation of Nuclei

Encapsulation of nuclei is performed essentially as described previouslyfor chromosome encapsulation. However, GMD size can be adjustment from25-35 to 45-55 μm to account for the larger size of nuclei bymodification of blade speed during the emulsification process.

Example 7 Improved Method of Chromosome Isolation

Chromosomes can be synchronized in metaphase by culturing cells fromwhich chromosomes are to be isolated with the flavonoid genisteintogether with colcemid, which is commonly used to block the cell-cycleat metaphase. This procedure yields a mitotic index of about 75%: (i.e.,three out of four cells yielded metaphase chromosomes).

Chromosomes are released from cells by hypotonic treatment. Quantitativerelease of chromosomes requires at least some physical shearing force,such as vortexing or passage through a gauge needle, and generate somecell debris. After chromosome release, nuclei are removed by low speedcentrifugation to remove nuclei. To remove proteins, chromosome lysatesare then dialyzed in a Slide-A-Lyzer™ (Pierce, Rockford, Ill.), whichhas a large pore membrane (100,000 molecular weight cut off), against a100 fold volume of CIB (with two changes). Two alternatives forseparating chromosomes from cellular debris can be employed.

In the first approach, chromosomes are captured with magnetic beadsconjugated to antibodies specific for the double stranded DNA of humanchromosomes. These antibodies are present in sera of patients sufferingfrom scleroderma (Chemicon International, Temecula, Calif.). Theimmunoglobulin fraction of the sera is isolated using protein A-agarose(Pierce, Rockford, Ill.). The IgG fraction is then biotinylated withbiotin-NHS ester (Molecular Probes, Eugene, Oreg.) using themanufacturer's procedure. Streptavidin magnetic microbeads (MiltenyiBiotec, Auburn, Calif.) are used to bind the biotinylated IgG fractionof the human sera from scleroderma patients. The microbeads are thenused to capture chromosomes. Chromosomes covered with magnetic beads areseparated from non-chromosomal material by means of a strong field MACSSeparator (Miltenyi Biotec). This approach also allows enrichment ofoccupied GMDs from the pool of mostly unoccupied GMDs.

In the second approach, cellular debris is separated from chromosomesusing antibodies specific for cytoskeletal proteins conjugated to asolid support. In the chromosome release step, buffer containingdetergent (Triton X-100) is used to solubilize membranes. Butcytoskeletal proteins, such as fibronectin filaments, actin filaments,or intermediate microfilaments, are mostly present in an insoluble form.Although most microtubule proteins are solubilized by colcemidtreatment, some remain. Antibodies against cytoskeletal proteins(fibronectin, actin, vimentin), are available from Accurate Chemical &Scientific (Westbury, N.Y.), Biosource International (Camarillo, Calif.)and Cytoskeleton (Denver, Colo.). These antibodies are conjugatedthrough reductive amination to AminoLink Plus Coupling agarose gel(Pierce, Rockford, Ill.). 0.5 ml of antibody-derivatized gel (in aCompact Reaction Column, United States Biochemical, Cleveland, Ohio) isused to capture insoluble fragments of cellular debris by passingdialyzed and concentrated chromosome solution through a column.

Example 8 Detection with Chemiluminescent Substrates

Exothermic chemical reactions generally release energy in the form ofvibrational or rotational excitation or heat. In chemiluminescentreactions, however, the electronically excited state is reached by achemical reaction and light rather than heat is generated. Theenergy-rich source in most chemiluminescent reactions is peroxide,hydroperoxide, 1,2 dioxetane, or dioxetane bonds. During the transitionof these excited intermediates to the electronic ground state, light isemitted in a process known as direct chemiluminescence. Novel acridaneor dioxetane chemiluminescent substrates, developed by Tropix, BioTecx,and collaborators at Lumigen allow detection of 10⁻¹⁹ moles ofhorseradish peroxidase or 10⁻²¹ moles of alkaline phosphatase (74-76), asubstantial improvement in sensitivity over fluorescence based detectionat 10⁻¹⁴ moles per liter.

Chemiluminescent substrates for alkaline phosphatase are currently allphenyl phosphate dioxetanes (PPD). PPD(4-methoxy-4-(3-phosphatephenyl)spiro[1,2dioxetane,-3,2′-adamantane]disodiumsalt) or CSPD® (chlorine derivative of PPD) are now widely used inclinical immunossays and protein and nucleic acid detection test kits.PPD or CSPD® based substrates also contain fluorescent enhancers whichpromote more efficient generation of chemiluminescent light throughextended glow kinetics.

Other substrates such as PS-1, a substrate for HRP which has just becomecommercially available, exploits Lumigen's discovery that esters ofN-alkylacridancarboxylic acid are efficiently oxidized by peroxidaseenzymes in the presence of hydrogen peroxide and a phenolic enhancer.The reaction, which requires only a minimal catalytic quantity of aperoxidase, converts the acridan compounds to the correspondingN-alkylacridinium ester. The PS-1 HRP substrate reaction produces anintense chemiluminescence, reaching a peak in approximately 10 minutes,with an extended decay over several hours. In a direct comparison withenhanced luminol based chemiluminescent reagents, previously the mostsensitive system for detecting HRP, PS-1 was shown to be 100 timesbrighter (74).

The current detection limit using fluorescence in situ hybridization(FISH) and non-isotopically-labeled probes is approximately 100 kb DNAtarget or 10-30 copies of mRNA (77-79). Although slightly strongersignals can be obtained using probes labeled with radioactive isotopes,unacceptably long exposure times of days to weeks are required to detectapproximately the same length DNA targets. Furthermore, health anddisposal concerns make this technology unsuitable for most laboratories.

In situ hybridization techniques have dramatically improved in recentyears both in terms of safety and sensitivity, primarily due to use ofenzymes instead of isotopes as reporter molecules. Due to high reactionturnover, horseradish peroxidase (HRP) or alkaline phosphatase (AP) arethe most frequently used reporter enzymes. Using reporter enzymes,localization of hybridization signals can be performed either withcolorimetric immunohistochemistry methods, which are less sensitive, orwith fluorescent methods, which increase sensitivity 10-100 fold, incomparison to the non-enzymatic methods (80).

The highest detection limits for both reporter enzymes are obtained,however, using chemiluminescent substrates which facilitate the emissionof light triggered by an enzymatic reaction. Approximate detectionlimits of several approaches are shown below:

Detection Method Detection Limit^(a) Base Pairs^(b) Color 10⁻¹⁰-10⁻¹²200-400 Fluorescence 10⁻¹³-10⁻¹⁵ 100-200 Radioisotopes 10⁻²⁰  10-100 PCR10⁻²² 0.4-10  Chemiluminescence 10⁻¹⁹-10⁻²¹  10-100 Microdrop CL- 10⁻²¹  10-100^(c) moles, kilobases, per each GMD

Tyramide Signal Amplification

The recently available Tyramide Signal Amplification (TSA) system (NENLife Sciences Products, Boston, Mass.) designed for fluorescence in situhybridization was used in order to compare amplified fluorescence signalwith chemiluminescence. TSA technology (80) uses horseradish peroxidase(HRP) to catalyze deposition of fluorophore or biotin labeled tyramidenear the site of the hybridized probe, proximal to the enzyme. HRP candeposit 10²-10³ tyramide molecules in 10 min, resulting in powerfulsignal amplification. In addition, since tyramide can be labeled bothwith fluorophores (TSA-Direct) and biotin (TSA-Indirect), bothfluorescent or chemiluminescent signal can be detected.

For fluorescent measurements after MISH, GMDs were incubated in TNBblocking buffer (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, 0.5% BlockingReagent, NEN) with diluted conjugates of HRP with streptavidin forbiotin labeled probes, or anti-fluorescein for fluorescein labeledprobes (both from NEN) for 30 min. After three subsequent washes withTNT (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, 0.05% TWEEN 20), GMDs wereincubated with 300 μl of diluted fluorescein-tyramide for 10 min.Unreacted tyramide was removed by washing GMDs twice with TNT. MISHsignals were then visualized using fluorescence microscopy.

Chemiluminescent Detection of Hybridized Probes

GMDs hybridized with biotin labeled probes were incubated for 30 min inTNB blocking buffer containing conjugate of either streptavidin-HRP(NEN) or HRP (Sigma) at dilutions of 1:100. GMDs hybridized withfluorescein-labeled probes were incubated with the conjugate ofanti-fluorescein-HRP (NEN) diluted 1:100. In some signal amplificationexperiments, prior to chemiluminescent measurements, tyramide labeledwith biotin was subsequently bound to streptavidin conjugate of eitherHRP or AP. Unreacted conjugates were removed by three successive washeswith TNT. GMDs were than pelleted by low speed centrifugation andresuspended in 0.05 ml 0.4×SSC. Aliquots diluted in a ratio of 1:5 withthe appropriate chemiluminescent substrate were than examined eitherusing a microscope equipped with a photon counting device or aluminometer.

For horseradish peroxidase detection, three Luminol-based and oneacridinium-based substrates were used. Two luminol-based substrates,LumiGLO™ and BM Chemiluminescence ELISA Substrate, were obtained fromKirkegaard and Perry Laboratories (KPL Inc., Gaithersburg, Md.) andBoehringer Mannheim Corp. (Indianapolis, Ind.), respectively. A thirdluminol-based substrate, NF-1, which does not require phenolicenhancers, was recently developed by BioTecx, Inc. (Houston, Tex.) andavailable for experimentation. The acridinium-based substrate, PS-1, wasobtained from Lumigen, Inc. (Southfield, Mich.).

For alkaline phosphatase detection, two adamantyl 1,2-dioxetane arylphosphate (PPD)-based substrates were used. Lumi-Phos 530, obtained fromLumigen, is a premixed formulation containing phenyl phosphatedioxetane, MgCl₂, cetyltrimethylammonium bromide, and an enhancer. CSPD,a derivative of PPD mixed with the Emerald II enhancer, was obtainedfrom Tropix, Inc. (Bedford, Mass.).

GMDs were incubated in substrate solutions for 5-10 min at roomtemperature for HRP or 10-15 min at 37° C. for AP. Light emitted fromGMDs soaked in chemiluminescent substrates was measured using anOPTOCOMP® I luminometer (MGM Instruments, Hamden, Conn.).

Derivatization of GMDs with HRP and AP

GMDs were formed from biotinylated agarose (FMC Bioproducts, Rockland,Me.) using standard emulsification procedures and sieved sequentiallythrough 62 and 45

m nylon mesh (Small Parts, Miami Lakes, Fla.) to obtain uniform sizemicrodrops. GMDs were then blocked with TNB blocking buffer for 15 min.Aliquots containing 5×10⁵ GMDs were reacted with appropriate dilutionsof streptavidin-HRP or streptavidin-AP (both from Sigma) for 30 min.After binding, GMDs were washed 3 times with TNT washing buffer.

Digital Image Microscopy

For imaging chemiluminescence, an Olympus BH-2 microscope, equipped withphase contrast objectives for visualizing GMDs, was connected to aphoton counting device through a C-mount. A Hamamatsu (Bridgewater,N.J.) C2400-32 ICCD (intensified CCD) camera was used as a photoncounting device. The camera consists of an image intensifier and a CCDcamera coupled with a relay lens and a control unit (Hamamatsu IIcontroller, model M4314). In this configuration, the CCD cameraeffectively visualizes 756×485 pixels (pixel size 8.4×9.8 μm). Imageswere created and modified using the Hamamatsu ARGUS 20 Image Processor,which permits real time image observation.

For fluorescence imaging, the microscope was connected to a HamamatsuC5985 cooled CCD camera and images were created using a Hamamatsucontroller and an Argus 20 Image Processor. The CCD camera was cooled to20° C. below the ambient temperature with a built in Peltier effectdevice. Both chemiluminescent and fluorescent images were analyzed andsuperimposed or pseudocolored using Adobe® Photoshop® 4.0 software(Adobe Systems, San Jose, Calif.) running on Windows® 95 operatingsystem.

Results

This research has demonstrated the feasibility of detecting at least2-10 kb DNA sequences located on single copy genes using probeshybridized to encapsulated nuclei and then imaged using microscopy and aCCD camera. A 2.2 kb DNA sequence of a single copy gene located on humanchromosome Y can be detected using a chemiluminescent substrate for HRP.This sequence was not detectable using fluorescently labeled probes.

We evaluated a variety of substrates for both HRP and AP reporterenzymes. Although the lowest detection limit for HRP, measured in aluminometer, was obtained using acridinium-based substrate, low lightlevel required for microscopic visualization destroyed the substrate.Two PPD (phenyl phosphate dioxetane) based substrates for AP were alsounaffected by red light required to focus specimens for microscopy.

To detect low chemiluminescent light levels we used several CCD cameras,including a Hamamatsu Peltier effect-cooled CCD and a Photometrics CH250cooled to −40° C. Low light levels generated from probe hybridizing tosingle copy gene sequences, which had approximately 4×10²-10³ moleculesof reporter enzyme, were best detected using photon counting devices. Bycomparison, about 10⁵ reporter molecules of enzyme bound to agarose GMDswere needed for detection using a cooled CCD camera, such as thePhotometrics CH250 (see Table 1 below).

TABLE 1 CCD camera detection limits for reporter enzyme moleculesconjugated to streptavidin. Reporter enzyme molecules CCD Cameradetected/GMD Hamamatsu C5985 CCCD >10⁵  Photometrics CH250 CCCD 10⁵Hamamatsu C2400-32 ICCD 10³

Example 9 RNA Detection in Encapsulated Cells

The ability rapidly to detect the presence of virus-specific orcancer-specific RNAs in individual cells present in low frequencies hasapplications in disease diagnosis, monitoring and treatment as well asblood screening. Microdrop in situ hybridization (MISII) is particularlysuited for these applications because it eliminates the need for cellfixation and prevents cell clumping which makes the detection of rarecells difficult. This examples describes detecting both cancer cells(positive for telomerase mRNA expression) and HIV-infected cells byusing a combination of MISH and flow cytometry.

Materials and Methods 1. Cell Lines and Culture Conditions

Human promyelocytic leukemia HL-60 cells leukemia were purchased fromAmerican Type Culture Collection (ATCC, Rockville, Md.). A3.01 cells andHIV infected cells H9/HTLV-III NIH 1983 were obtained from the NIH AIDSResearch and Reference Reagent Program, Rockville, Md. All cells werecultured in RPMI 1640 medium supplemented with 10% fetal bovine serum(FBS) and were grown at 37° C. in the presence of 5% CO₂.

2. Probes and Fluorescent Labeling

Oligonucleotide probes for detecting RNA labeled with fluorescein at 5′end were purchased from Oligoes Etc., Wilsonville, Oreg. Oligos labeledwith horseradish peroxidase were purchased from Biosorce International,Camarillo, Calif. Oligonucleotide probes for detection of HIV RNA arederived from gag region of HIV genome. Sequences of oligonucleotideprobes are depicted below:

HIV RNA detection H1 5′-(HRP)CCA TTC TGC AGC TTC CTC ATT GAT GGT CTC-3′H2 5′-(HRP)CTT GTC TTA TGT CCA GAA TGC TGG TAG GGC-3′ Telomerase mRNAdetection BF-1 5′FITC-CCA ACA AGA AAT CAT CCA CCA AAC GCA GGA GC 3′ BF-35′FITC-GAG GCT GTT CAC CTG CAA ATC CAG AAA CAG 3′ BF-4 5′FITC-GAA GGTTTT CCC GTG GGT GAG GTG AGG TG 3′

3. Cell Encapsulation

Human cells harvested from various stages of growth were pelleted by lowspeed centrifugation, washed with Hanks Balanced Salt Solution (HBSS)containing 0.1% diethylpyrocarbonate (DEPC), and resuspended in the samebuffer at concentrations 20×10⁶ cells per ml. For encapsulation ofcells, pluronic acid was used as a surfactant because it does not affectcell membrane integrity during emulsification.

A cell-agarose mixture (2.3% Type XII agarose and 0.1% pluronic acid inIIBSS containing 2×10⁶ cells per 0.52 ml) was prepared by melting 0.4 mlagarose in HBSS at 100° C., cooling the agarose to 57° C., and adding100 μl of cell suspension and 20 μl of 10% pluronic acid. The mixturewas held at 65° C. for 5 min and then quickly added dropwise to 15 ml ofCelMix^(T) 200 emulsion matrix (One Cell Systems, Cambridge, Mass.)pre-warmed to 6° C. Gel microdrops were created using a CellSys100^(T)Microdrop Maker (One Cell Systems, Cambridge, Mass.) equipped with a 1.6cm blade using successive rotor speeds of 2,200 rpm for 1 min at 20-25°C., 2,200 rpm for 1 min at 0° C., and 1,200 rpm for 7 min at 0° C. TheGMDs were then separated from the emulsion matrix by centrifugation at400×g for 7 min. The pellet containing encapsulated cells was washedtwice with DEPC treated HBSS and stored at 4° C. in the same buffer andused within a week. However, encapsulated cells can be stored long-term(at least for a month) in 70% ethanol at −20° C.

4. Microdrop In Situ Hybridization (MISH) of Encapsulated Human Cells

All solutions used for cellular RNA detection were prepared in DEPCtreated water. 1 μl of DEPC (10% in 70% ethylalcohol) was added toapproximately 100,000 GMDs containing 10,000 cell-occupied GMDs in avolume of 50 μl. After a 15 min incubation at room temperature, an equalvolume of 2× hybridization buffer was added and the mixture wasincubated for a given time at temperatures ranging from 50 to 58° C. Forhybridization with the oligo probes, 2× hybridization solutioncontained, 1.2 M Tris-IIC1, pH 8.0, 0.1 mg/ml salmon sperm DNA, 0.1mg/ml E. coli tRNA, 100 units of placental RNAsc inhibitor/ml and 200 μlof Vanadyl Ribonucleoside Complex/ml (BRL, Bethesda, Md.). Before mixingwith encapsulated cells 10-20 picomoles/0.1 ml of oligonucleotide probeswere added to 2× hybridization buffer. After performing hybridization at55° C., GMDs were washed using wash buffer (WB, 0.05 M Tris-IIC1, pH0.0, 0.15 M NaCl, 0.2 mM EDTA, 0.1% Tween 20) at temperatures rangingfrom 20 to 55° C. Hybridization signals were amplified by a TyramideSignal Amplification (TSA, NEN^(T) Life Science Products, Boston, Mass.)either directly (with HRP labeled probes) or after binding withanti-fluorescein-HRP (with fluorescein-labeled probes).

To detect HIV RNA with oligo probes directly labeled with HRP,encapsulated cells were first washed three times with HRP stabilizingbuffer (Biotecx, Cambridge, Mass.) before adding 2× hybridizationbuffer.

5. Tyramide Signal Amplification

The Tyramide Signal Amplification (TSA) system (NEN Life SciencesProducts, Boston, Mass.) is designed for non-isotopic in situhybridization. TSA technology (37) uses horseradish peroxidase (HRP) tocatalyze deposition of tyramide labeled with fluorophores near the siteof the hybridized probe, proximal to the enzyme. HRP can deposit 10²-10³tyramide molecules in 10 min, resulting in powerful signalamplification. After MISH, GMDs were incubated in TNB blocking buffer(0.1M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% Blocking Reagent, NEN) with1:200 diluted conjugate of anti-fluorescein HRP (NEN) for 30 min. Afterthree subsequent washes with TNT (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl,0.05% Tween 20), GMDs were incubated with 1:100 dilutedtyramide-fluorescein for 10 min. Unreacted tyramide was removed bywashing GMDs twice with TNT.

6. Microscopy and Digital Image Analysis

The integrity of encapsulated cellular nucleic acids (both DNA and RNaafter fluorescent staining with 0.2 μg/ml acridine orange) and MISHfluorescence signals were examined visually using an Olympus BH-2microscope (phase contrast 40× SPlan 0.4 objective) equipped forepifluorescence with appropriate filters for DAPI, fluorescein orphycoerythrin. Digital images were taken with a cooled CCD camera (B/WHamamatsu C5985-02 with control unit) connected to the microscope.Digital images were processed with the assistance of computer software(Adobe Photoshop version 4.0, Adobe Systems, San Jose, Calif.).

7. Flow Cytometric Analysis of Encapsulated Cells after MISH

Flow cytometric analysis after MISH was performed using an EPICSElite^(T) (Coulter Corporation, Miami, Fla.). 15 mW of a 488 nm line ofan air cooled argon laser was used to excite the fluorescein inhybridized probes. The trigger parameter for collecting events wasforward scatter (detecting encapsulated cells). 10,000 list mode eventswere collected at a rate of approximately 600 occupied GMDs per secondand analyzed using elite 4.01 software.

Results

FIG. 7 shows flow cytometric detection of gag HIV RNA in encapsulatedHIV infected cells after hybridization with two HRP-labeled oligoprobes. A2.01 cells with the same lineage as H9 cells were used ascontrol cells. Both control and HIV infected cells were vultured for 4days. The peaks are color coded and represent the following: Red peak,A3.01 cells, probes not present (mean fluorescence 0.258); Blue peak, H9HTLVIII cells, probes not present (mean fluorescence=0.289); Yellowpeak, A3.01 cells, probes present (mean fluoresce ce=3.41); Green peak,H9HTI.VIII cells, probes present (mean fluorescence—19.2). Meanfluorescence of HIV infected and non-infected cells hybridized witholigo probes was used for the calculation of S/N value, which was foundto be 5. It has previously been estimated that H9/HTLV-III cells expressapproximately 250-500 copies of HIV-specific RNA. Detection of such alow copy RNA number indicates the sensitivity of the assay for detectionof HIV-infected cells in human blood.

FIG. 8 shows detection of telomerase mRNA in HL-60 (model cancer cellline) and human PBMCs using fluorescein-labeled oligonucleotide probesfollowed by TSA signal amplification. Hybridization conditions and TSAamplification are described in Materials and Methods. The histograms arecolor coded as follows: red=HL-60 cells; black=human PBMCs. Left peakrepresents unoccupied GMDs and right peak represents GMDs occupied withsingle cells. The relative mean fluorescence of the right peaks in 220for HL-60 cells and 78 for human PBMCs (S/N—2.8). These results show thepower of this methodology for detecting differential expression of mRNAin different cell types, and particularly between normal and cancercells.

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While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1-35. (canceled)
 36. A method of clinical analysis comprising:encapsulating biological entities in gel microdrops, wherein saidbiological entities are from a tissue sample from a patient; storing themicrodrops for at least an hour; and performing a clinical analysis onsaid encapsulated biological entities.
 37. The method of claim 36,wherein the microdrops are stored for at least six months.
 38. Themethod of claim 36, wherein the clinical analysis is performed onchromosomes from the microdrops.
 39. The method of claim 36, wherein theclinical analysis comprises contacting the microdrops with a probe thatis complementary to a nucleic acid molecule, the presence of which isindicative of a disease state and; diagnosing the existence or prognosisof the disease from the hybridization or lack of hybridization of theprobe to a nucleic acid molecule within an encapsulated cell.
 40. Themethod of claim 39, wherein the disease is a disease associated with agenetic defect.
 41. The method of claim 39, wherein the disease iscancer.
 42. The method of claim 39, wherein the probe is complementaryto at an exonic segment of a gene.
 43. The method of claim 39, whereinthe probe is complementary to at least two successive exons of a geneand specifically hybridizes to RNA expressed from the gene.
 44. Themethod of claim 39, wherein the disease comprises the presence of avirus.
 45. The method of claim 39, wherein the virus is a retrovirus.46. A method of storing a biological entity in a gel microdrop,comprising: forming a population of gel microdrops encapsulating apopulation of biological entities within a gel matrix; and storing atleast a portion of the population of gel microdrops under conditions topreserve the biological entities intact.
 47. The method of claim 46,wherein the biological entities comprise cells, viruses, organelles,mitochondria, chloroplasts, nuclei, chromosomes, proteins, nucleicacids, or fragments thereof.
 48. The method of claim 46, wherein thepopulation of gel microdrops is stored for at least one hour, for atleast one day, for at least one week, for at least one month, for atleast six months, for at least one year, for at least two years, or forat least five years.
 49. The method of claim 46, wherein the gelmicrodrop comprises agarose.
 50. The method of claim 46, wherein atleast a portion of the population of gel microdrops encapsulate a singlebiological entity.
 51. The method of claim 50, wherein the biologicalentity comprises a nucleic acid hybridized to a probe comprising acomplementary sequence prior to storage.
 52. The method of claim 46,further comprising releasing the gel microdrop encapsulated biologicalentity by digesting the gel matrix.
 53. A method of storing a patientsample in a gel microdrop, comprising: obtaining a biological samplefrom a patient; forming a population of gel microdrops encapsulating thebiological sample within a gel matrix; and storing at least a portion ofthe population of gel microdrops under conditions to preserve thepatient sample intact and without degradation.
 54. The method of claim53, wherein the biological sample from the patient comprises one or morecells taken from a tissue or blood sample.
 55. The method of claim 54,wherein the one or more cells are propagated prior to encapsulationwithin the population of gel microdrops.
 56. The method of claim 54,wherein the tissue sample is obtained from a location within the patientsuspected of comprising a genetic abnormality or an indicia of suspecteddisease.
 57. The method of claim 56, wherein the suspected disease is acancer, an autoimmune disease, or a disease of the central nervoussystem.
 58. The method of claim 53, wherein the population of gelmicrodrops is stored for at least one hour, for at least one day, for atleast one week, for at least one month, for at least six months, for atleast one year, for at least two years, or for at least five years. 59.The method of claim 53, wherein at least a portion of the population ofgel microdrops encapsulate a single biological entity from the sample,the biological entity comprising a cell, a chromosome, or a nucleicacid.
 60. The method of claim 53, further comprising releasing the gelmicrodrop encapsulated biological sample by digesting the gel matrix.